Project description:Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet (HFD)-induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex 1 (mTORC1) and AMP-activated protein kinase (AMPK), are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol-3-kinase (PI3K) and mTORC2, kinases upstream of AKT and important for its activation, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WD repeat domain 24 (WDR24), the GATOR2 component essential for the Sestrin2–GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2–mTORC2 interaction, completely ablated Sestrin2’s effect on AKT activation. We also noted that Sestrin2 directly binds to the pleckstrin homology (PH) domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2.
Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter. We took a systematic approach to examine the genome-wide distribution (using ChIP-chip) of the various Mediator subunits. Mediator occupancy was also assayed in mutants for most of the CTD kinases and in strains where some CTD serines had been replaced by alanines. Mediator ChIPs were performed with Myc-tagged subunits, except in some preliminary experiments where polyclonal antibodies were used. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In some of the preliminary experiments, non immunoprecipitated DNA (input) was used as the control. In addition to Mediator ChIPs, the project includes TFIIB and RNAPII (Rpb3) ChIP-chip datasets. All ChIP-chip experiments were done in duplicates except for the preliminary experiments that were done in monoplicat for the most part. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
Project description:The diets of industrialized countries reflect the increasing use of processed foods, often with the inclusion of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent. Xanthan gum’s chemical structure is distinct from the host and dietary polysaccharides that are more commonly expected to transit the gastrointestinal tract, and little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears contingent on a single uncultured bacterium in the family Ruminococcaceae. Our data reveal that this primary degrader cleaves the xanthan gum backbone before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides intestinalis that is incapable of consuming polymeric xanthan gum but grows on oligosaccharide products generated by the Ruminococcaceae. Feeding xanthan gum to germfree mice colonized with a human microbiota containing the uncultured Ruminococcaceae supports the idea that this additive can drive expansion of this primary degrader along with exogenously introduced Bacteroides intestinalis. Our work demonstrates the existence of a potential xanthan gum food chain involving at least two members of different phyla of gut bacteria and provides an initial framework to understand how widespread consumption of a recently introduced food additive influences human microbiomes.
Project description:Transcribed CGG repeat expansions cause the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). CGG repeat RNAs sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic peptides in the cytoplasm. To identify proteins involved in these processes, we employed a CGG repeat RNA-tagging system to capture repeat associated RBPs by mass spectrometry in mammalian cells. We identified several SR (serine/arginine-rich domain) proteins that interact selectively with CGG repeats basally and under cellular stress. These same proteins modify toxicity in a Drosophila model of FXTAS. Genetic or pharmacological targeting of serine/arginine protein kinases (SRPKs) inhibits RAN translation in cells and toxicity in both FXTAS and C9orf72 ALS/FTD model flies. Furthermore, SRPK inhibitors suppressed CGG repeat toxicity in rodent neurons. These findings demonstrate roles for CGG repeat RNA binding proteins in repeat toxicity and support further evaluation of SRPK inhibitors in modulating RAN translation associated with repeat expansion disorders.
Project description:The aim of the experiment was to compare chromatin states between healthy and hyperglycaemic mice. We focused on dendritic cells extracted from the lungs. Nuclei were extracted from 200.000 sorted dendritic cells per sample. H3K27me3 and H3K27ac were then profiled using CUT&RUN protocol.
Project description:Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain. In order to examine the genome-wide localization of RNAPII and Spt6 in Saccharomyces cerevisiae, RNAPII and Spt6 along with associated DNA sequences were immunoprecipitated using anti-8WG16 and anti-HA antibodies, respectively. The RNAPII and Spt6 chromatin immunoprecipitation was performed in duplicate from WT cells as described below. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.
Project description:Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. We have shown that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. Here, we explore the interactome of EZH2 and of a phosphodeficient mutant EZH2_T367A. We identified novel interactors of EZH2, and identified interactions that are dependent on the phosphorylation and cellular localization of EZH2 that may play a role in EZH2 dependent metastatic progression.
Project description:We performed m6A-RIPs in Ascl1-induced neurons (iNeurons) to investigate the neuronal m6A epitranscriptome. Immunoprecipitation was done twice using two different antibodies, acquired from Abcam and Synaptic Systems (SySy), allowing for a more robust detection of m6A modification marks. Additionally, RIP-seq was performed separately with intact and fragmented RNA. The former approach allowed to identify proportions of m6A-modified transcripts among the total number, while the latter approach provided the information to identify genomic coordinates of m6A peaks.
Project description:α-1-antitrypsin (AAT) regulates protease activity in the lungs and liver. The presence of one or more copies of a mutant Z allele (AAT (E342K), called ATZ), results in low serum levels of ATZ along with intracellular inclusions of polymeric forms, causing lung emphysema and liver cirrhosis. Our experiments show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media to cell ratio. This effect is more specific for ATZ compared with AAT, and is only partially dependent on the glycan-binding site of CRT. The CRT-related chaperone calnexin (CNX) does not promote enhanced ATZ secretory trafficking, despite the higher cellular abundance of CNX-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body (IB) formation, and reducing ATZ interactions with components required for ER-Golgi trafficking. These findings indicate a novel role for CRT in promoting the secretory trafficking of a polymerogenic protein. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ IBs.
Project description:Small RNAs exert an effect through diverse RNA interference pathways to transcriptionally or post-transcriptionally silence their targets. The Piwi-interacting RNAs (piRNAs) represent a germline-specific small RNA pathway where Piwi proteins themselves are thought to mediate piRNA biosynthesis. Here, we provide strong evidence for a piRNA amplification loop in zebrafish, in which Ziwi and Zili bind piRNAs of opposite polarity. Furthermore, we describe a function for Zili in transposon defense and germ cell differentiation, as well as a crucial function in meiosis, significantly extending the function of Piwi proteins beyond the control of transposable elements in vertebrates. small RNA from total gonads or Argonaute IPs were cloned and sequenced using 454 GS FLX system.